Improving the Thermostability of a Fungal GH11 Xylanase via Fusion of a Submodule (C2) from Hyperthermophilic CBM9_1-2

Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure’s stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.


Introduction
Carbohydrate degradation involves a series of hydrolases, especially xylanase (EC 3.2.1.8) [1]. Xylanase catalyzes the hydrolysis of xylan, a major constituent of hemicelluloses. The enzyme has sparked renewed interest due to its industrial applications, including the paper and pulp sectors as well as the food and feed industries [2]. The application value of xylanases in industrial processes depend on their thermal stability and activity. In some procedures, such as production drying, feed pelleting, maltification, etc., xylanases with excellent thermal stability to adapt high-temperature environments are highly demanded [3]. However, most natural xylanases belong to mesophilic enzymes [4]. Although several xylanases have been extracted from thermophilic microorganisms, their expression levels and enzymatic activities are insufficient for industrial use [5]. Thus, many projects have been undertaken to develop xylanases with improved thermostability and activity to create novel enzymes withstanding harsh conditions [3,[6][7][8].
Most xylanases from different species are categorized into glycoside hydrolase (GH) families 10 and 11, with the remainder belonging to families 5,8,30, and 43 based on the sequence similarities in the catalytic domain [9]. GH11 xylanases, in comparison to GH10 xylanases, have received significant attention because of their small scale, strict substrate

Effects of the C2 Submodule on Enzyme Activity and Kinetic Characterization
The degradation activities of four xylanases (CDBFV, C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) on Corncob xylan, Birch xylan, Bagasse xylan, Avicel, and CMC-Na were determined at pH 5.5 and 60.0 °C. The results showed that the four xylanases had the best degradation activity on Corncob xylan and Birch xylan, followed by bagasse xylan, and almost did not degrade Avicel and CMC-Na (Supplementary Materials Table S2). On different substrates, there were no significant differences in the degradation activities of the four xylanases, suggesting that the C2 submodule had no impact on CDBFV substrate specificity. The binding ability of four xylanases to insoluble substrates (insoluble Corncob xylan, Bagasse xylan, Avicel, and CMC-Na) was measured at pH 5.5 and 60.0 °C. The

Effects of the C2 Submodule on Enzyme Activity and Kinetic Characterization
The degradation activities of four xylanases (CDBFV, C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) on Corncob xylan, Birch xylan, Bagasse xylan, Avicel, and CMC-Na were determined at pH 5.5 and 60.0 • C. The results showed that the four xylanases had the best degradation activity on Corncob xylan and Birch xylan, followed by bagasse xylan, and almost did not degrade Avicel and CMC-Na (Supplementary Materials Table S2). On different substrates, there were no significant differences in the degradation activities of the four xylanases, suggesting that the C2 submodule had no impact on CDBFV substrate specificity. The binding ability of four xylanases to insoluble substrates (insoluble Corncob xylan, Bagasse xylan, Avicel, and CMC-Na) was measured at pH 5.5 and 60.0 • C. The results showed that the four xylanases had no strong binding ability to insoluble substrates.
After reacting at pH 5.50 and 60.0 • C for 2.0 h, the protein binding rates were less than 20.0% (Supplementary Materials Figure S1). The addition of a C2 substructure did not improve the binding of CDBFV to insoluble substrates. Compared to CDBFV, the activities of C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 against the Corncob xylan were greater by 18.0-28.0%, and the K M values were slightly lower (Table 1). These results indicate that the domain C2 submodule is beneficial for increasing the enzymatic activity of CDBFV.

Effects of the C2 Submodule on pH and Temperature Characterization
The optimal pH of the four xylanases was determined within the pH range from 3.0 to 9.0. According to the pH-activity curve (Supplementary Materials Figure S2a), the maximal activity was observed at pH 5.5. Four xylanases displayed high stability in the pH range of 4.0-9.0 for 60.0 min, according to the pH stability curve (Supplementary Materials Figure S2b), and kept roughly 80.0% of their original activity. The results show that the pH characteristics of CDBFV are unaffected by the C2 submodule.

Molecular Modeling and Structural Analysis
To explore the specific reasons for the difference in the thermal stability of wild-type CDBFV between the N-terminal (C2-CDBFV) and C-terminal (CDBFV-C2) connections of the C2 submodule, the trRosetta server was employed based on the energy-minimized structure algorithm and 15,000 amino acid multiple sequence alignment modeling. Among them, the wild-type CDBFV and C2 submodule exhibit the highest structural consistency with xylanase from N. patriciarum (PDB ID: 3WP4) and xylanase 10A of T. maritima (PDB ID: 1I8U_A), respectively. The two chimeric xylanases were predicted from the zero-folded protein structure by trRosetta. Five structural models were obtained for each chimera. A modeler was used to fill in the atoms that may be missing in the structure of each obtained model. SAVES V6.0 was used to evaluate all 3D models, and Ramachandran plots of these models were obtained (Supplementary Materials Figure S3). In the best 3D protein models selected, more than 85% of the residue was within the permissible

Molecular Modeling and Structural Analysis
To explore the specific reasons for the difference in the thermal stability of wild-type CDBFV between the N-terminal (C2-CDBFV) and C-terminal (CDBFV-C2) connections of the C2 submodule, the trRosetta server was employed based on the energy-minimized structure algorithm and 15,000 amino acid multiple sequence alignment modeling. Among them, the wild-type CDBFV and C2 submodule exhibit the highest structural consistency with xylanase from N. patriciarum (PDB ID: 3WP4) and xylanase 10A of T. maritima (PDB ID: 1I8U_A), respectively. The two chimeric xylanases were predicted from the zero-folded protein structure by trRosetta. Five structural models were obtained for each chimera. A modeler was used to fill in the atoms that may be missing in the structure of each obtained model. SAVES V6.0 was used to evaluate all 3D models, and Ramachandran plots of these models were obtained (Supplementary Materials Figure S3). In the best 3D protein models selected, more than 85% of the residue was within the permissible range, indicating that the chimeric structures predicted by trRosetta from the zero-fold protein structure were highly reliable and could be followed up for structural analysis. The obtained model was imported into PyMOL to compare the 3D structure; it was clearly found that there are obvious differences in the structure of chimeric xylanases C2-CDBFV and CDBFV-C2. The protein structure of chimeric xylanase C2-CDBFV is more slender, and the distance between submodule C2 and CDBFV is relatively long (Supplementary Materials Figure  S4a). However, the chimeric xylanase CDBFV-C2 is the opposite. The linker is inserted into the β-barrel cavity of CDBFV, and protein interaction can be obviously observed between submodule C2 and CDBFV (Figure 3a).
analyzed the hydrogen bond interaction between the three parts of the chimeric xylanase C2-CDBFV, and we found that there is no interaction between the submodule C2 and CDBFV. Only two additional hydrogen bonds were formed between the linker and CDBFV, namely, E240-K267 and V244-S260, respectively (Supplementary Materials Figure S4b). Then, we further analyzed the hydrogen bond interaction between the three parts of the chimeric xylanase CDBFV-C2, and we found that all three parts interacted. Three additional hydrogen bonds were formed between the linker and CDBFV, namely, V263-G260, V277-T59, and S101-G276, respectively (Figure 3b). Up to five additional hydrogen bonds were formed between submodule C2 and CDBFV, namely, S41-K463, S42-N455, S44-G371, S42-S462, and G61-K369, respectively (Figure 3c). In brief, by adding submodule C2 and a linker to the N-terminus of CDBFV, the chimeric xylanase C2-CDBFV forms two additional hydrogen bonds compared to wild-type CDBFV. By adding submodule C2 and a linker to the C-terminus of CDBFV, the chimeric xylanase CDBFV-C2 forms eight additional hydrogen bonds compared to wild-type CDBFV. The main reason the thermal stability of the chimera xylanase CDBFV-C2 is better than that of C2-CDBFV is that more new and additional hydrogen bonds are formed.

MD Simulation Details of the Chimeric CDBFV-C2
To explore the protein structural stability of the chimera CDBFV-C2 with the best thermal stability, a 30.0 ns molecular dynamics (MD) simulation was performed on the chimeric xylanase CDBFV-C2 and its components. Generally speaking, the root means square deviation (RMSD) values of the chimeric CDBFV-C2 and its components-CDBFV, Linker, and C2 substructures-are all less than 0.7 nm (Figure 4a). Adding C2 to CDBFV To further explore the specific interaction of the different connection directions of submodule C2 on the protein structure of CDBFV, we explored the protein structure of the hydrogen bond network between the submodule C2, linker, and CDBFV. First, we analyzed the hydrogen bond interaction between the three parts of the chimeric xylanase C2-CDBFV, and we found that there is no interaction between the submodule C2 and CDBFV. Only two additional hydrogen bonds were formed between the linker and CDBFV, namely, E240-K267 and V244-S260, respectively (Supplementary Materials Figure S4b). Then, we further analyzed the hydrogen bond interaction between the three parts of the chimeric xylanase CDBFV-C2, and we found that all three parts interacted. Three additional hydrogen bonds were formed between the linker and CDBFV, namely, V263-G260, V277-T59, and S101-G276, respectively (Figure 3b). Up to five additional hydrogen bonds were formed between submodule C2 and CDBFV, namely, S41-K463, S42-N455, S44-G371, S42-S462, and G61-K369, respectively (Figure 3c). In brief, by adding submodule C2 and a linker to the N-terminus of CDBFV, the chimeric xylanase C2-CDBFV forms two additional hydrogen bonds compared to wild-type CDBFV. By adding submodule C2 and a linker to the C-terminus of CDBFV, the chimeric xylanase CDBFV-C2 forms eight additional hydrogen bonds compared to wild-type CDBFV. The main reason the thermal stability of the chimera xylanase CDBFV-C2 is better than that of C2-CDBFV is that more new and additional hydrogen bonds are formed.

MD Simulation Details of the Chimeric CDBFV-C2
To explore the protein structural stability of the chimera CDBFV-C2 with the best thermal stability, a 30.0 ns molecular dynamics (MD) simulation was performed on the chimeric xylanase CDBFV-C2 and its components. Generally speaking, the root means square deviation (RMSD) values of the chimeric CDBFV-C2 and its components-CDBFV, Linker, and C2 substructures-are all less than 0.7 nm (Figure 4a). Adding C2 to CDBFV did not cause huge structural differences in C2 and CDBFV parts. In Figure 4a, the structural alterations of C2 and CDBFV were evaluated by RMSD values with black and red lines. It is found that the RMSD values of C2 and CDBFV during 30.0 ns MD simulation were smaller than 0.5 nm, indicating that structures of C2 and CDBFV did not experience huge configurational alteration. This indicates that the protein structure of the chimeric xylanase CDBFV-C2 is relatively stable. Local plasticity of the chimeric xylanase CDBFV-C2 was evaluated by the root mean square fluctuation (RMSF) value of the C α atom, which was calculated using MD simulation data for the last 30.0 ns of the initial structure (Figure 4b). Generally, except for the larger RMSF value of the N-and C-terminal parts, the RMSF value of each amino acid residue of the chimeric xylanase CDBFV-C2 as a whole is less than 0.6 nm, with an average value of 0.2 nm (Figure 4b). The results show that the protein structure of the chimeric xylanase CDBFV-C2 has strong rigidity. In short, the chimeric xylanase CDBFV-C2 has a highly rigid and stable protein structure.

Discussion
The development of thermostable xylanase is critical for meeting practical industrial demands [5]. To obtain a xylanase chimera with enhanced thermostability, the thermostable domain C2 submodule was first fused to the N-terminus, C-terminus, and both termini of CDBFV in this work. As a result, the chimeric xylanase with the C-terminal C2 submodule has higher thermostability, and fusion at the C-terminus is more effective than fusion at the N-terminus. Through structural analysis, it was determined that the mechanism for the improved thermal stability of xylanase CDBFV-C2 is that S42-S462, T59-E277, S41-K463, and S44-G371 can form stable hydrogen bonds, which were beneficial to increase the stability of the CDBFV-C2 protein structure. Thus, several points are worth discussing.
First, the C2 submodule, in particular, improved the thermostability of xylanase. In the previous study, this effect was investigated by dividing the CBM9_1-2 module into two smaller parts, C1 and C2, which were transplanted into Xyn to create the chimeras To gain insight into the improved thermostability of the chimeric xylanase CDBFV-C2, the stability of eight additional hydrogen bonds was monitored during the simulation (Figure 4c). During the 30.0 ns MD simulation, hydrogen bonding interactions formed between the amino acid residues S42-S462 and T59-E277 were 82.8 and 62.7 percent, respectively, have a greater than 50.0% probability of forming stable interactions (Figure 4c), while S41-K463 and S44-G371 amino acid residues have a nearly 30.0% probability of forming stable hydrogen bonds. S42-N455, G276-S101, G61-K369, and G260-V263, on the other hand, have a chance of forming stable hydrogen bonds of less than 10.0% (Figure 4c). Even G61-K369 and G260-V263 cannot form stable hydrogen bonds. In addition, the RMSF values of the key amino acid residues of the newly formed four additional hydrogen bonds were counted. The RMSF values of the amino acid residues involved in forming four stable additional hydrogen bonds are all fewer than the average, which can be evaluated in Figure 4d. Analysis of MD simulations reflects that the amino acid residues involved in forming four additional hydrogen bonds improve the stability of the chimeric xylanase CDBFV-C2 protein structure. In short, the improved heat resistance of the chimeric xylanase CDBFV-C2 may be due to the ability of S42-S462, T59-E277, S41-K463, and S44-G371 to form stable additional hydrogen bonds, which is beneficial in increasing the stability of the CDBFV-C2 protein structure and thus significantly improving its thermal stability.

Discussion
The development of thermostable xylanase is critical for meeting practical industrial demands [5]. To obtain a xylanase chimera with enhanced thermostability, the thermostable domain C2 submodule was first fused to the N-terminus, C-terminus, and both termini of CDBFV in this work. As a result, the chimeric xylanase with the C-terminal C2 submodule has higher thermostability, and fusion at the C-terminus is more effective than fusion at the N-terminus. Through structural analysis, it was determined that the mechanism for the improved thermal stability of xylanase CDBFV-C2 is that S42-S462, T59-E277, S41-K463, and S44-G371 can form stable hydrogen bonds, which were beneficial to increase the stability of the CDBFV-C2 protein structure. Thus, several points are worth discussing.
First, the C2 submodule, in particular, improved the thermostability of xylanase. In the previous study, this effect was investigated by dividing the CBM9_1-2 module into two smaller parts, C1 and C2, which were transplanted into Xyn to create the chimeras Xyn-C1 and Xyn-C2. The Xyn-C2 thermal inactivation half-life (69.3 min) was four or five times longer than that of Xyn (17.6 min), Xyn-C1 (13.4 min), and the original chimera containing CBM9_1-2 (13.8 min) [1,[19][20][21]. C2 submodule applied to the N, C, and both sides of CDBFV improved the thermal stability of xylanase by about 1.5, 5.0, and 3.7 times, respectively, compared to wild-type xylanase CDBFV (Figure 2). Previous reports showed that the chimerization of submodule C2 with the N-terminus of A. niger xylanase could increase its thermal stability by about four times compared with wild-type xylanase, which is consistent with our conclusions [1]. However, the relationship between CBMs and stability remains controversial. The following three conclusions have been reported: (1) CBMs can improve the thermal stability and optimal temperature of the chimeric enzyme [3,15]; (2) the removal and addition of CBMs have no effect on the thermal stability of the chimeric enzyme [13]; (3) the addition of CBMs reduces the thermal stability of the chimeric enzyme [12,14,16]. The stability of the chimeric enzyme is related not only to its own amino acid sequence but also to the different properties of CBMs. Choosing a CBM derived from a more thermally stable enzyme for chimerization may yield a higher probability of success.
Second, adding CBM to wild enzymes can be performed via various methods. The possibilities include (1) adding CBM to the N-terminus of the wild enzyme [3,12,22]; (2) adding CBM to the C-terminus of the wild enzyme [1,16]; (3) adding CBM to both sides of the wild enzyme [13,15]; (4) adding multiple CBMs to one side of the wild enzyme [23]; (5) replacing or deleting the CBM in the wild enzyme [16]. These results show that different methods of adding CBM have different effects on wild enzymes. However, we cannot predict which way of adding is best in advance. In this work, we found that the Cterminal chimeric module C2 submodule of CDBFV improves thermal stability considerably more than the N-terminal. The thermostable recombinant expands the opportunities for modifying the C-terminal region of GH11 xylanases. Then, the thermal stability of the N-terminal chimeric module C2 sub-module is not significantly improved (Figure 2). The CDBFV's N-terminal structure has a longer "tail," which causes the xylanase CDBFV and the module C2 sub-module to be further apart in space [24]. The structure has little influence on each other. The chimeric xylanase C2-CDBFV is also more slender, and the distance between submodule C2 and CDBFV is relatively long, according to our modeling structure (Figure 3a). Simultaneously, the fourth amino acid residue (Cys) at the N end of the CDBFV forms a pair of disulfide bonds with the 172nd amino acid residue (Cys) [24], aiding in the stabilization of the CDBFV's N-terminal structure. In summary, it is impossible to determine which of the wild enzyme proteins is most suitable for adding CBM, but it can be determined that the side with the most impact on the structure of the wild enzyme protein will produce better outcomes.
Third, another exciting aspect of this study is that fusing the thermostable module C2 submodule to the N-terminus, C-terminus, and both termini of CDBFV increased the activity of CDBFV. Compared with wild-type CDBFV, the specific activities of the chimeric xylanase added to the N-, C-, and both termini of CDBFV increased by 17.8, 18.7, and 27.9%, respectively ( Table 1). The same conclusion was reported in a study of chimeras between A. niger xylanase and the module C2 submodule [1]. In addition, we also found that the N-terminal (C2-CDBFV) and C-terminal (CDBFV-C2) have no significant difference regarding the improvement of enzyme activity. There is no positional effect, such as thermal stability (Table 1). One possible reason for this is that the two residues Trp175 and Trp71 in the module C2 submodule accomplish a "hand" function [25]. This allows the chimeric enzyme to "unaffected by position, actively grab more substrates" instead of randomly colliding with substrates [1,25]. The enzyme activity of xylanase C2-CDBFV-C2 is the highest among the three chimeras, which may be due to the cumulative effect of the C2 submodule on the increase in enzyme activity [1]. Unfortunately, we did not find that chimeric enzymes improve the efficiency of insoluble substrate hydrolysis (Supplementary Material Figure S1) and substrate specificity (Supplementary Material  Table S2). This is different from the result that the C2 submodule and A. niger xylanase can improve the binding efficiency of insoluble substrates [1]. The specific reasons may be: (1) the structural similarity between xylanase CDBFV and A. niger xylanase is only 38.0%, and their structures and properties are very different, leading to differences between the two results [25,26]; (2) the C2 submodule belongs to the C-type CBM. It only binds to monosaccharides, oligosaccharides, or polysaccharide terminal glycosyls, without grooves, and the binding sites are relatively concentrated, which are easy to embed in the space structure and cannot be completely exposed [25].
Finally, the linker peptide provides the necessary space for each domain to form an active conformation, and the appropriate linker is an important factor that affects the folding of the domains [17,[27][28][29][30]. The designed linker peptide will interfere with the thermostability [27][28][29] and enzymatic activity [17,27] of adjacent domains. As previously reported, the flexibility of the linker region is of great significance for enzyme activity [30][31][32], so we used a natural 22-residue linker peptide (PEVLPPLPKESRISEGEAVVVG) to connect these domains. The linker we selected contains four Pro and two Ser residues, which can form flexible hinge regions and aid in creating an active spatial conformation between different domains [1,25]. This may be one of the important reasons why the C2 submodule helps to improve the thermostability and activity of xylanase XynCDBFV.

Expression and Purification of the Chimeric CDBFV Variants
Transformed cells were grown in LB medium at 37.0 • C until the OD 600 nm reached 0.7, and xylanases were induced by adding IPTG to a final concentration of 0.5 mM. After further incubation for 15.0 h at 20.0 • C, supernatants were harvested after the cells were disrupted by high-pressure cell breakage (Constant Systems 8TANT, Daventry, Northants, UK) and centrifugation at 13,000 rpm/min for 20.0 min at 4.0 • C. After removing cell debris, both the N-terminal and C-terminal His-tagged enzymes containing the supernatant were purified by affinity chromatography using a Ni-NTA agarose column. Afterward, the purified xylanases were detected using 12.0% polyacrylamide SDS-PAGE analysis, and the enzyme concentration was measured by a Bradford protein assay kit. The gene fragment size and protein molecular mass of the chimeras were calculated by DNAMAN v6 [1,16,18].

Assay Properties of the Chimeric CDBFV Variants
Xylanases' standard activity was measured using the 3,5-dinitrosalicylic acid (DNS) method [3]. The amount of enzyme consumed by producing 1.0 µmol of xylose per min at 60.0 • C and pH 5.5 (100.0 mM, citrate phosphate buffer) was defined as one unit of xylanase activity. To measure xylanase activity, the reaction system was configured as follows: 450.0 µL of citrate phosphate buffer (100.0 mM, pH 5.5) was mixed with 450.0 µL of the substrate stock solution (1.0% m/v) of Corncob xylan (Yuanye, Shanghai, China) and 100 µL of diluted enzyme solution. After reacting for 10.0 min at 60.0 • C, 1500.0 µL of DNS was added to terminate the reaction, and the reaction mixture was then boiled in a water bath for 5.0 min. After the reaction solution was cooled to room temperature, a SpectraMax (Bio-Rad, Hercules, CA, USA) in a 540.0 nm reader was used to measure the absorbance of the solution supernatant. The blank group reaction without enzyme was performed similarly to the negative control. All measurements were conducted in triplicate.
The enzymatic activity of CDBFV was assayed in parallel with the variants C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 for assessment of properties. The optimal pH for the purified xylanases activity was determined with buffer pH values from 3.0 to 9.0 at 37.0 • C. The enzymatic stabilities in various pH ranges were judged by measuring the residual enzymatic activities after incubating the enzymatic solution at different buffer pH values from 3.0 to 11.0 at 37.0 • C for 60.0 min. The pH ranges of the buffers were as follows: citrate phosphate buffer (3.0-8.0), Tris-HCl (7.0-9.0), and glycine-NaOH (9.0-12.0). The optimal temperature of purified xylanases was measured within the range from 10.0 to 80.0 • C at pH 5.5. Thermostability was determined at pH 5.5 and 60.0 • C after preincubating the enzyme for different times at 60.0, 65.0, or 70.0 • C. Xylanase-specific activity was assayed on sodium carboxymethyl cellulose (CMC-Na), Avicel, Beechwood xylan, Bagasse xylan, and Corncob xylan (Yuanye, Shanghai, China). The binding ability of the insoluble substrates was analyzed by measuring the protein binding rate in the reaction system after the enzyme solution was reacted at 60.0 • C for 2.0 h [33]. The K M and V max values for purified xylanases were determined using 0.0%-1.0% Corncob xylan as the substrate in pH 5.5 citrate phosphate buffer at 60.0 • C. The data were fit based on the classic Michaelis-Menton function.

Molecular Modeling and Structural Analysis
The crystal structures of CDBFV from N. patriciarum (PDB ID: 3WP4) [26] and C2 from xylanase 10A of T. maritima (PDB ID: 1I8U_A) [25] were used as templates to model the binding modules of CDBFV, C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2. The procedure for constructing the three-dimensional structure for all xylanases was performed via the trRosetta online website (https://yanglab.nankai.edu.cn/trRosetta/, accessed on 14 September 2021) according to the methods described previously [24,34,35]. The obtained models were performed using Modeller to complement possible missing atoms in the structure and selection of the best models via the online site SAVES v6.0 [24,34,35]. The obtained models were compared with the visualization software PyMOL, and the molecular effects in the structure were displayed and analyzed [36,37].

MD Simulation Details of the Chimeric CDBFV-C2
The structure of the chimera xylanase CDBFV-C2 was built by trRosetta server [24,34,35]. The MD simulation system was performed via GROMACS program suite version 4.5.7 and Amber ffff99SB force field [38,39]. Sodium or chloride ions (100.0 mM) were added to the system to neutralize the charge [40]. Normal MD simulations were performed in an isothermal-isobaric ensemble for 30.0 ns, which was solvated with TIP3P waters in an octahedral box [41], and the minimal distance between each protein and edge of the box was set to 0.8 nm [42]. Before the simulation, we performed 1000-step energy minimization and equilibrated for 5 ns in NPT ensemble by restraining all heavy atoms [43].
Hydrogen bonds between amino acid residues in the simulation system were analyzed by using g_hbond in the GROMACS suite. Geometrical criteria, which include donoracceptor distance (≤0.4 nm) and hydrogen-donor-acceptor angle (≤30.0 • ), are used to calculate hydrogen bond. For each time frame, if both the donor-acceptor distance and the hydrogen-donor-acceptor angle satisfy the criteria, the number of hydrogen bonds will be counted as 1.0, and 0.0 otherwise. The number of hydrogen bonds was calculated based on the whole 30.0 ns simulation (30,000 frames in total) in the system, and the error bar represents one standard error based on the averaged number of hydrogen bonds every 100.0 ps in each system.

Conclusions
This research shows that the xylanase from N. patriciarum and the N-and C-termini (both termini of which fuse a C2 submodule from a hyperthermophilic CBM9_1-2) can enhance thermostability. Compared with wild-type xylanase, the half-lives of the three chimeras increased by about 1.5, 5.0, and 3.7 times, respectively. Structural analysis showed that the thermal stability of the chimera CDBFV-C2 with the best thermal stability was significantly improved by the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which is beneficial to increasing the stability of the CDBFV-C2 protein structure, thereby significantly improving its thermal stability.